Introduction into Cell Biology 2 The building blocks of life - Proteins

1. Introduction into Cell Biology 2The building blocks of life - Proteins

2. Intro into cell biology 2 Molecular Organisation of a cell

3. Fig. 1.7

4. Building Blocks of Life -> Different Shapes

6. Proteins Amino Acids are linked by peptide bonds

7. 20 Natural Occurring Amino Acids are divided into groups according to their side chains:

8. The Aromatic Amino Acids

10. Cys can cross-link between 2 polypeptide chains -> Disulfide bridge

11. Proteins are Polypeptides Direction of a Protein

12. 3D Structure of Proteins

13. Interactions between side chains and backbone -> Fold of a protein (3D structure)

14. Noncovalent interactions within and between biological molecules

15. Secondary Structure: 1. α – Helix 2. β-Strands -> β-Sheets 3. Loops and Turns

16. α-Helix

17. α-helical coiled coil proteins: Form superhelix Found in myosin, tropomyosin (muscle), fibrin (blood clots), keratin (hair) Examples of α-Helical Proteins: Hair

18. Examples of α-Helical Proteins: Muscle α-helical coiled coil proteins: Form superhelix Found in myosin, tropomyosin (muscle), fibrin (blood clots), keratin (hair)

19. Examples of α-Helical Proteins:

20. β-Strands -> β-sheets

21. Examples of β-sheet Proteins: Fatty acid binding protein -> β barrels structure OmpX: E. coli porin Antibodies

24. Turns and Loops: Loops in Receptors Turn

25. Tertiary Structure: 3D structure of a polypeptide chain

26. Quaternary Structure: Polypeptide chains assemble into multisubunit structures Cell-surface receptor CD4 Tetramer of hemoglobin

27. Protein Folding Folding is a highly cooperative process (all or none) Folding by stabilization of Intermediates

28. Protein Folding by Chaperons

29. Misfolded protein -> Alzheimer Protein fibrillation

30. Function of Proteins

31. Molecular Machines – Transcription Initiation Complex

32. Function of Proteins Specific binding of ligands -> Immunoglobins

33. Function of Proteins Conformational change of lactoferrin upon binding of Fe Conformational change induced by Calcium

34. Function of Proteins Activation by modification GFP fluorescent: Rearrangement and oxidation of Ser-Tyr-Gly

35. Function of Proteins Model of enzymatic reaction mechanism

36. Proteins Key properties: • Proteins are linear polymersbuilt of Amino Acids • Proteins contain many functional groups (i.e.. side chain of AA) • Proteins interact with proteins and with other biological molecules to form complexes • Proteins can bind and/or modify other molecules • Proteins can be rigid or can have regions with high flexibility

37. Enzyme Kinetics • Enzymes DO NOT shift the equilibria but enhance the rates of the reactions (lower the activiation energy!!!)

38. X‡ intermediate H Reaction coordinate Transition state • Unstable state of maximum energy • Not an intermediate • Metastable state • Intermediates are species that appear in a reaction mechanism but not in the overall balanced equation. DH‡ H DH0 Reaction coordinate

40. rate = D[A] D[B] rate = - Dt Dt Reaction Kinetics Thermodynamics – does a reaction take place? Kinetics – how fast does a reaction proceed? Reaction rate is the change in the concentration of a reactant or a product with time (M/s). A B D[A] = change in concentration of A over time period Dt D[B] = change in concentration of B over time period Dt Because [A] decreases with time, D[A] is negative. 13.1

41. A B time rate = D[A] D[B] rate = - Dt Dt 13.1

42. Suppose an enzyme were to react with a substrate, giving a product. Basic problem of enzyme kinetics S + E P + E If we simply applied the law of mass action to this reaction, the rate of reaction would be a linearly increasing function of [S]. As [S] gets very big, so would the reaction rate. This doesn’t happen. In reality, the reaction rate saturates.

43. Michaelis and Menten In 1913, Michaelis and Menten proposed the following mechanism for a saturating reaction rate k1 k2 S + E ES P + E k-1 Complex. product

44. Michaelis-Menten Kinetics • When [S] << KM, the reaction increases linearly with [S]; I.e. vo = (Vmax / KM ) [S] • Very little [ES] is formed • When [S] = KM, vo = Vmax /2 (half maximal velocity); this is a definition of KM: the concentration of substrate which gives ½ of Vmax. This means that low values of KM imply the enzyme achieves maximal catalytic efficiency at low [S]. • When [S] >> Km, vo = Vmax Where activity measurements should be performed: 1. [S] very high 2. all enzyme bound in [ES] complex

45. Michaelis-Menten Kinetics When the enzyme is saturated with substrate, the reaction is progressing at its maximal velocity, Vmax. Combing the steady-state assumption (d[ES]/dt=0) with the conservation condition ([E]T=[E] + [ES]) vo leads to the Michaelis-Menten Equation of enzyme kinetics: where Km is KM= (k-1 + k2)/k1

46. Michaelis-Menten Kinetics What is Vmax and KM ? • KM gives an idea of the range of [S] at which a reaction will occur. The larger the KM, the WEAKER the binding affinity of enzyme for substrate. • Vmax gives an idea of how fast the reaction can occur under ideal circumstances.

47. Michaelis-Menten Kinetics Determination of Enzyme kinetics -> Measure activity (velocity) at different substrate concentrations Determine activity of an Enzyme -> Measure at substrate concentration of above 10KM -> no substrate limitation [E]T=[ES]

48. Michaelis-Menten Kinetics How to measure activity of an enzyme using photometrical method ? Lambert Beer law: A= c ε l Where A is the absorbance c is the concentration (mol/L) ε is the molar absorption coefficient (L/mol cm) l is the path length of sample (cm) Rate = activity = Δc/Δt -> ΔA/Δt = l εΔc/Δt activity = rate (unit) -> Δc/Δt = (ΔA/Δt)/lε Definition: 1 Unit of an enzyme will catalyse the reaction of 1 μmol of substrate within 1 min at certian pH and Temperature. Measure ΔA/Δt (change in absorption/min) for a special enzyme and a high substrate concentration -> from that you can calculate activity of that enzyme at special Temp., solvent, pH, pressure.

49. Michaelis-Menten Kinetics How do determine experimentally KM and Vmax ? (y= d + k x) Lineweaver-Burk plot Eadie-Hofstee plot